Little is known about the functions of the many different unconventional myosins in vivo. Most cells express many myosin family members, each in a distinct subcellular distribution. Their relative contributions to cellular organization, dynamics, and function remains unclear. In our previous studies we have identified the first myosin of class VI (Drosophila 95F myosin) and have shown that it is an actin-based cytoplasmic transporter involved in membrane remodeling during the syncytial blastoderm stage of development. Subsequently, we have identified myosin VI mutants that affect spermatogenesis. Phenotypic studies of these mutants suggest that myosin VI is a transporter during membrane remodeling in this differentiation process as well. Myosin VI may also be important for facilitating interactions between the actin and microtubule cytoskeletons. We discovered that myosin VI and CLIP (cytoplasmic linker protein-190), a microtubule- binding protein, are associated, as judged by biochemical and immunolocalization criteria. Both of these proteins have been implicated in intracellular transport. The two proteins are associated in several processes in which both actin-based and microtubule-based processes are thought to be important. Two of these are neuronal transport and anchoring of RNPs in early embryos. We hypothesize that the myosin VI-CLIP interaction facilitates movement of transported components from one cytoskeletal system to the other. To test our hypotheses about myosin VI function, our studies will focus on membrane remodeling and transport roles for myosin VI in three cell types: spermatids, where we will use primarily genetic techniques to identify interacting proteins; neuronal cells, where we will use biochemical fractionation and imaging of motility in vivo; and the early embryo, where we will investigate interactions important for RNP localization using dominant interfering molecules and antibody inhibition approaches. CLIP's role in myosin VI mediated-processes will be analyzed using dominant interfering molecules expressed in vivo. We will examine the biochemical mechanism of myosin VI's participation in these processes using experiments in vitro that map the domains important for interactions with other proteins and define the biochemical activities associated with complexes containing myosin VI. Since it is thought that unconventional myosins in other organisms play roles similar to those we propose for myosin VI in Drosophila, our experiments will reveal important information about myosin function and associations that should be generally applicable.